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Reduced Affinity (reduced + affinity)

Distribution by Scientific Domains

Life Sciences	30%
Chemistry	30%

Selected Abstracts

ChemInform Abstract: Synthesis and Biological Evaluation of New 4-Arylpiperidines and 4-Aryl-4-piperidinols: Dual Na+ and Ca2+ Channel Blockers with Reduced Affinity for Dopamine D2 Receptors.

CHEMINFORM, Issue 15 2002
Hirokazu Annoura
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source]

Cloning and paratope analysis of an antibody fragment, a rational approach for the design of a PAI-1 inhibitor

JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2 2004
K. Verbeke
Summary., This study reports the cloning, characterization and paratope analysis of the plasminogen activator inhibitor-1 (PAI-1) neutralizing single-chain variable fragment 56A7C10 (scFv-56A7C10). ScFv-56A7C10-wt exhibits a similar affinity (KA = 1.01 ± 0.3 × 109 m,1) and PAI-1 inhibitory capacity (90 ± 6% PAI-1 inhibition at a 16-fold molar excess and IC50 = 44 ± 14 ng mL,1) as MA-56A7C10 (KA = 1.43 ± 0.4 × 109 m,1, 90 ± 2% PAI-1 inhibition at a 16-fold molar excess and IC50 = 122 ± 26 ng mL,1). Subsequently, alanine scanning of the six complementarity determining regions (CDRs) was performed and the scFv-56A7C10-mutants (n = 26) were analyzed for their PAI-1 binding and PAI-1 inhibitory properties. Mutation of the residues Y32 and V33 in the CDR1 of the heavy chain (HCDR1) and the residues R98, H99, W100 or F100a (HCDR3) resulted in reduced PAI-1 inhibitory capacities (IC50 , 418 ng mL,1), confirmed by reduced affinities (14-, 17-, 7-, 9- and 16-fold reduced, respectively, vs. scFv-56A7C10-wt). In the light chain, mutation of the residues W50 (LCDR2), H91, Y92, D93, or W96 (LCDR3) resulted in reduced PAI-1 inhibitory properties (IC50 , 160 ng mL,1) and decreased affinities (i.e. 4-, 9-, 3-, 3- and 2-fold reduced affinity, respectively, vs. scFv-56A7C10-wt). Furthermore, an overlapping peptide scan confirmed the importance of the HCDR3 region. These data, combined with a three-dimensional model of scFv-56A7C10, reveal the molecular and structural properties of the paratope and contribute to the rational design of PAI-1 neutralizing compounds. [source]

Molecular analysis of the A322D mutation in the GABAA receptor ,1 -subunit causing juvenile myoclonic epilepsy

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2005
Klaus Krampfl
Abstract Juvenile myoclonic epilepsy (JME) belongs to the most common forms of hereditary epilepsy, the idiopathic generalized epilepsies. Although the mode of inheritance is usually complex, mutations in single genes have been shown to cause the disease in some families with autosomal dominant inheritance. The first mutation in a multigeneration JME family has been recently found in the ,1 -subunit of the GABAA receptor (GABRA1), predicting the single amino acid substitution A322D. We further characterized the functional consequences of this mutation by coexpressing ,1 -, ,2 - and ,2 -subunits in human embryonic kidney (HEK293) cells. By using an ultrafast application system, mutant receptors have shown reduced macroscopic current amplitudes at saturating GABA concentrations and a highly reduced affinity to GABA compared to the wild-type (WT). Dose,response curves for current amplitudes, activation kinetics, and GABA-dependent desensitization parameters showed a parallel shift towards 30- to 40-fold higher GABA concentrations. Both deactivation and resensitization kinetics were considerably accelerated in mutant channels. In addition, mutant receptors labelled with enhanced green fluorescent protein (EGFP) were not integrated in the cell membrane, in contrast to WT receptors. Therefore, the A322D mutation leads to a severe loss-of-function of the human GABAA receptor by several mechanisms, including reduced surface expression, reduced GABA-sensitivity, and accelerated deactivation. These molecular defects could decrease and shorten the resulting inhibitory postsynaptic currents (IPSCs) in vivo, which can induce a hyperexcitability of the postsynaptic membrane and explain the occurrence of epileptic seizures. [source]

Site-directed mutagenesis and footprinting analysis of the interaction of the sunflower KNOX protein HAKN1 with DNA

FEBS JOURNAL, Issue 1 2005
Mariana F. Tioni
The interaction of the homeodomain of the sunflower KNOX protein HAKN1 with DNA was studied by site-directed mutagenesis, hydroxyl radical footprinting and missing nucleoside experiments. Binding of HAKN1 to different oligonucleotides indicated that HAKN1 prefers the sequence TGACA (TGTCA), with changes within the GAC core more profoundly affecting the interaction. Footprinting and missing nucleoside experiments using hydroxyl radical cleavage of DNA showed that HAKN1 interacts with a 6-bp region of the strand carrying the GAC core, covering the core and nucleotides towards the 3, end. On the other strand, protection was observed along an 8-bp region, comprising two additional nucleotides complementary to those preceding the core. Changes in the residue present at position 50 produced proteins with different specificities. An I50S mutant showed a preference for TGACT, while the presence of lysine shifted the preference to TGACC, suggesting that residue 50 interacts with nucleotide(s) 3, to GAC. Mutation of Lys54,Val produced a protein with reduced affinity and relaxed specificity, able to recognize the sequence TGAAA, while the conservative change of Arg55,Lys completely abolished binding to DNA. Based on these results, we propose a model for the interaction of HAKN1 with DNA in which helix III of the homeodomain accommodates along the major groove with Arg55, Asn51, Lys54 and Ile50, establishing specific contacts with bases of the GACA sequence or their complements. This model can be extended to other KNOX proteins given the conservation of these amino acids in all members of the family. [source]

Characterization of the NAD+ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site-directed mutagenesis

FEBS JOURNAL, Issue 22 2000
Nikolas E. Labrou
The 2,,3,-dialdehyde derivative of ADP (oADP) has been shown to be an affinity label for the NAD+ binding site of recombinant Candida boidinii formate dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biphasic pseudo first-order saturation kinetics. The rate of inactivation exhibited a nonlinear dependence on the concentration of oADP, which can be described by reversible binding of reagent to the enzyme (Kd = 0.46 mm for the fast phase, 0.45 mm for the slow phase) prior to the irreversible reaction, with maximum rate constants of 0.012 and 0.007 min,1 for the fast and slow phases, respectively. Inactivation of formate dehydrogenase by oADP resulted in the formation of an enzyme,oADP product, a process that was reversed after dialysis or after treatment with 2-mercaptoethanol (> 90% reactivation). The reactivation of the enzyme by 2-mercaptoethanol was prevented if the enzyme,oADP complex was previously reduced by NaBH4, suggesting that the reaction product was a stable Schiff's base. Protection from inactivation was afforded by nucleotides (NAD+, NADH and ADP) demonstrating the specificity of the reaction. When the enzyme was completely inactivated, approximately 1 mol of [14C]oADP per mol of subunit was incorporated. Cleavage of [14C]oADP-modified enzyme with trypsin and subsequent separation of peptides by RP-HPLC gave only one radioactive peak. Amino-acid sequencing of the radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific reaction at the nucleotide binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Molecular modelling studies were used to create a model of C. boidinii FDH, based on the known structure of the Pseudomonas enzyme, using the modeller 4 program. The model confirmed that Lys360 is positioned at the NAD+ -binding site. Site-directed mutagenesis was used in dissecting the structure and functional role of Lys360. The mutant Lys360,Ala enzyme exhibited unchanged kcat and Km values for formate but showed reduced affinity for NAD+. The molecular model was used to help interpret these biochemical data concerning the Lys360,Ala enzyme. The data are discussed in terms of engineering coenzyme specificity. [source]

The interaction between ,S, the stationary phase , factor, and the core enzyme of Escherichia coli RNA polymerase

GENES TO CELLS, Issue 3 2002
Frédéric Colland
Background: The RNA polymerase holoenzyme of Escherichia coli is composed of a core enzyme (subunit structure ,2,,,) associated with one of the , subunits, required for promoter recognition. Different , factors compete for core binding. Among the seven , factors present in E. coli, ,70 controls gene transcription during the exponential phase, whereas ,S regulates the transcription of genes in the stationary phase or in response to different stresses. Using labelled ,S and ,70, we compared the affinities of both , factors for core binding and investigated the structural changes in the different subunits involved in the formation of the holoenzymes. Results: Using native polyacrylamide gel electrophoresis, we demonstrate that ,S binds to the core enzyme with fivefold reduced affinity compared to ,70. Using iron chelate protein footprinting, we show that the core enzyme significantly reduces polypeptide backbone solvent accessibility in regions 1.1, 2.5, 3.1 and 3.2 of ,S, while increasing the accessibility in region 4.1 of ,S. We have also analysed the positioning of ,S on the holoenzyme by the proximity-dependent protein cleavage method using ,S derivatives in which FeBABE was tethered to single cysteine residues at nine different positions. Protein cutting patterns are observed on the , and ,, subunits, but not ,. Regions 2.5, 3.1 and 3.2 of ,S are close to both , and ,, subunits, in agreement with iron chelate protein footprinting data. Conclusions: A comparison between these results using ,S and previous data from ,70 indicates similar contact patterns on the core subunits and similar characteristic changes associated with holoenzyme formation, despite striking differences in the accessibility of regions 4.1 and 4.2. [source]

NSF binds calcium to regulate its interaction with AMPA receptor subunit GluR2

JOURNAL OF NEUROCHEMISTRY, Issue 6 2007
Jonathan G. Hanley
Abstract N -ethylmaleimide-sensitive fusion protein (NSF) is essential for numerous Ca2+ -triggered vesicle trafficking events. It functions as a molecular chaperone to regulate trafficking protein complexes such as the soluble NSF attachment protein (SNAP) receptor complex and the ,-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-protein interacting with C-kinase (PICK1) complex. AMPAR trafficking is fundamental to processes of synaptic plasticity, which may underlie learning and memory. Changes in synaptic strength brought about by AMPAR trafficking are triggered by a post-synaptic influx of Ca2+, which may have numerous molecular targets including PICK1. NSF binds AMPAR subunit glutamate receptor subunit 2 (GluR2) and functions to maintain receptors at the synapse. In this study, it was showed that NSF is a Ca2+ -binding protein and that GluR2,NSF interactions are inhibited by the presence of 15 ,mol/L Ca2+. NSF Ca2+ -binding is reciprocally inhibited by the presence of GluR2 C-terminus. Mutant of NSF that binds Ca2+ with reduced affinity and binds GluR2 with reduced sensitivity to Ca2+ was identied. In addition, the interaction of ,SNAP with PICK1 is sensitive to Ca2+. This study demonstrates that the GluR2-NSF-,SNAP-PICK1 complex is regulated directly by Ca2+, allowing for the transduction of Ca2+ signals into concerted alterations in protein,protein interactions to bring about changes in AMPAR trafficking during synaptic plasticity. [source]

Reactive site-dependent phenotypic alterations in plasminogen activator inhibitor-1 transgenic mice

JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2007
M. EREN
Summary.,Background:,Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of plasminogen activators (PAs) and plays a role in the regulation of a number of physiological processes including the degradation of extracellular matrix proteins, cell proliferation and migration, and intracellular signaling. Aim:,To characterize the effects of durable expression of a stable form of human PAI-1 and to characterize important structure,function relationships in PAI-1 in vivo.Methods:,We developed transgenic mice lines overexpressing stable variants of human PAI-1 under the control of the murine preproendothelin-1 promoter and characterized the phenotypic alterations displayed by transgenic mice. Results:,Transgenic mice expressing an active form of human PAI-1 (PAI-1-stab) display complex phenotypic abnormalities including alopecia and hepatosplenomegaly. Reactive site mutant transgenic mice expressing inactive PAI-1 exhibit complete phenotypic rescue, while transgenic mice expressing PAI-1 with reduced affinity for vitronectin manifest all of the phenotypic abnormalities present in PAI-1-stab transgenic mice. Conclusions:,The protease inhibitory activity of PAI-1 toward PAs and/or other serine proteases is necessary and sufficient to promote complex phenotypic abnormalities and mediates many of the physiological effects of PAI-1 in vivo. [source]

Characterization of an immunologic polymorphism (D79H) in the heavy chain of factor V

JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2004
M. Van Der Neut Kolfschoten
Summary.,Background: During the study of a family with hereditary factor (F)V deficiency (FV Amersfoort, 1102 A > T in exon 7) we identified an individual with 5% FV heavy chain antigen (FVHC) and 50% FV light chain antigen (FVLC). Further testing revealed that apart from the FV Amersfoort allele a second variant FV allele was segregating in this family, which encodes for a FV molecule with a reduced affinity for mAb V-23 used in the FV heavy chain ELISA (ELISAHC). Objective: Identification and characterization of the molecular basis responsible for the reduced affinity of the variant FV for mAb V-23. Methods: Family members of the proband were screened for mutations in the exons coding for the heavy chain of FV, after which the recombinant variant FV could be generated and characterized. Next, the cases and controls of the Leiden Thrombophilia Study (LETS) were genotyped for carriership of the variant FV. Results: In the variant FV allele a polymorphism in exon 3 (409G > C) was identified, which predicts the replacement of aspartic acid 79 by histidin (D79H). Introduction of this mutation in recombinant FV confirmed that it reduces the affinity for binding to mAb V-23. The substitution has no effect on FV(a) stability and Xa-cofactor activity. In Caucasians the frequency of the FV-79H allele is ,5%. Analysis of the LETS revealed that the FV-79H allele is not associated with FV levels (FVLC), activated protein C sensitivity (using an activated partial thromboplastin time-based test) or risk of venous thrombosis (OR 1.07, CI 95: 0.7,1.7). Conclusion: The D79H substitution in FV should be considered as a neutral polymorphism. The monoclonal antibody V-23, which has a strongly reduced affinity for FV-79H, is not suitable for application in diagnostic tests. [source]